WO2019096096A1

WO2019096096A1 - Multi-arm targeting conjugate

Info

Publication number: WO2019096096A1
Application number: PCT/CN2018/115059
Authority: WO
Inventors: 袁建栋; 黄仰青; 宋云松
Original assignee: 博瑞生物医药（苏州）股份有限公司
Priority date: 2017-11-14
Filing date: 2018-11-12
Publication date: 2019-05-23

Abstract

Disclosed is an anti-cancer targeting conjugate modified by a multi-arm polymer, wherein modification by a water-soluble polymer can enhance the water solubility of the conjugate and increase a drug loading amount; when the targeting molecule is LRKLRKRLLLRKLRKRLL, the conjugate can increase brain targeting so as to make same pass through the blood brain barrier more easily, and can play the role of a brain-targeting peptide, allowing same to be at a higher concentration in a target tissue; and when the targeting molecule is folic acid, the conjugate can actively target tumor cells in which folate receptors are abundantly expressed, and better exert anti-tumor effects and enhance the targeting property.

Description

Multi-arm targeting conjugate

Technical field

The present invention generally relates to multi-arm targeting conjugates. In particular, the present invention relates to multi-arm PEG-modified targeted anti-cancer conjugates, and more particularly, the present invention is a conjugate of a brain-targeting molecule linked to an anti-cancer drug by a multi-arm PEG.

Background technique

WO2005028539, WO2010019233, WO2011063156, WO2011063158 disclose a clinical phase III drug nktr 102, which is mainly used for metastatic breast cancer, developed by Nektar Therapeutics. The drug is a water-soluble multi-branched polymer prodrug to increase the drug loading, and the structure is as follows:

The compound is linked to irinotecan using a multi-arm PEG to increase water solubility, increase drug loading, and reduce side effects without changing the anticancer effect. However, the drug still has disadvantages, for example, poor targeting, can not act on specific cancer cells, while killing cancer cells, it also affects the performance of normal cells, and the incidence of adverse reactions is still relatively high.

Apolipoprotein E (APOE gene, apoE protein) is a 34 kD glycoprotein composed of a 10 kD lipid-binding domain and a 22 kD receptor domain (binding to the low-density lipoprotein family of receptors). Endogenous apoE or intraventricular injection of apoE whole protein plays a neuroprotective role by down-regulating the central nervous system inflammation, reducing oxidative stress, anti-excitatory amino acid toxicity, and directly increasing neurotrophic nutrition.

However, due to the large molecular weight of intact apoE, it cannot pass the blood-brain barrier under normal conditions, which limits its application as an exogenous drug. The synthetic apoE peptidomim retains most of the natural three-dimensional structure of the receptor binding domain, retains most of the properties of the whole protein, competes with the apoE whole protein for receptor binding sites on the surface of macrophages, and initiates a signaling cascade. To play its neuroprotective role.

LRKLRKRLLLRKLRKRLL is an apoE peptidomimetic that acts as a brain targeting peptide through the blood-brain barrier. Its structure is as follows:

The folate receptor (FR) is a glycosylated phosphatidylinositol-linked membrane glycoprotein, and folate (FA) can specifically bind to folate receptors. The addition of folic acid to the complex allows folate to bind to the folate receptor on the tumor cells, so that the complex can be taken up into the cell by cell receptor-mediated endocytosis. This provides a specific targeted drug delivery route for folate receptor-positive tumor cells. Due to this stable binding of folic acid to folate receptors, folic acid is now widely used as a targeting system for various targeted drug delivery.

Summary of the invention

The present invention discloses three novel targeted multi-arm drug conjugates, which are compound a, compound b, and compound c represented by formula (I), formula (II), and formula (III), respectively:

To further illustrate the inventive concept of the present invention, the above conjugate can be expressed as formula (IV):

Where R is the organic center, ie in the structure of the conjugate

Represents the junction of atoms. Starting from the central carbon atom of the organic center, four branches are emitted, each of which is identical. Each branch consists of a polymer POLY, a multivalent linker L, a targeting molecule T, and an active agent D.

The polymer POLY is polyethylene glycol, and in the present invention, it is specifically:

n is 113,

Representing the junction of an atom, the oxygen atom labeled "&" is an atom attached to the organic center "R".

It should be understood by those skilled in the art that in the field of polymers, n represents the degree of polymerization of the polymer, that is, the average number of repeating units contained in the polymer macromolecular chain, depending on the molecular weight of the polymer. For example, when n is 113, it means that the average value is 113.

The multivalent linker L of compound a is:

The multivalent linker L of compound b is:

The multivalent linker L of compound c is:

The symbol "*" represents the point of attachment of the multivalent linker L to the target molecule T by "cysteine", "#" represents the point of attachment of the multivalent linker L to the active agent D, and "%" represents the link of the multivalent linker L and POLY. Junction.

The targeting molecule T of compound a is apoE peptidomimetic LRKLRKRLLLRKLRKRLL;

The target molecule T of compound b and compound c is folic acid, and the folic acid structure is as follows:

The active agent D of compound a, compound b and compound c are all irinotecan, and the structure of irinotecan is as follows:

The present invention is a multi-arm polymer modified targeted anti-cancer conjugate, wherein the water-soluble polymer modification enhances the water solubility of the conjugate and increases the drug loading; the targeting molecule LRKLRKRLLLRKLRKRLL increases brain targeting The conjugate is more likely to pass the blood-brain barrier and exert the function of brain-targeting peptide to make it higher in the target tissue; the target molecule is folic acid, and folic acid acts as a targeting molecule to actively target the expression of folate receptor. Rich tumor cells, better anti-tumor efficacy, increased targeting, so that the concentration of the conjugate in the target tissue is higher.

L is an arbitrary linker, which firstly connects the targeting molecule with the anticancer drug, and then connects the "targeting molecule, the anticancer drug and the polymer arm, so that the whole conjugate forms an organic whole. The conjugate of the present invention is a typical prodrug, and the active agent D is released by hydrolysis or enzymatic action, and is separated from the mother to exert physiological activity.

The conjugates of the present invention exhibit high loading capacity so that the total dose can be lowered to treat a particular disease, such as cancer. That is, the conjugate active agent carrier of the present invention is capable of covalently bonding to the active agent molecule, allowing a greater amount of therapeutic dosage form (i.e., active agent moiety) to be administered per certain amount of conjugate. The modification of the conjugate of the present invention by the water-soluble polymer is essentially that the conjugate is also hydrophilic, and in particular, when the active agent is a water-insoluble drug, the bioavailability of the conjugate is improved.

The conjugates of the invention are capable of exhibiting a stronger effect than tissues that are not coupled, and are more enriched in the human or other animal tissues.

The conjugate drug precursors of the present invention contain a number of unique properties, especially where the active agent is an anti-cancer compound. This prodrug inhibits tumor growth with higher efficiency. The small molecule we use is a small molecule known to have anticancer properties. However, by combining with a multi-branched polymer as described above, its efficacy and pharmacokinetics are greatly improved compared to the small molecule (e.g., the anticancer compound itself).

The conjugates of the present invention include pharmaceutically acceptable salts including inorganic salts and organic salts, and typical salts include nitrates, sulfates, phosphates, hydrofluoric acid salts, hydrochlorides, hydrobromides, hydroiodides. , formate, lactate, benzoate, acetate, trifluoroacetate, dichloroacetate, trichloroacetate, mixed chlorofluoroacetate, citrate, oxalic acid Salts, sulfonates, methanesulfonates, triflate, heptanesulfonate and the like, of which trifluoroacetate and heptanesulfonate are preferred.

Compound a typical trifluoroacetate salt comprises from one to forty molecules of trifluoroacetate. Preferably, each branch is bound to a conjugate of twelve molecules of trifluoroacetate, the preferred conjugate being forty-eight molecules of trifluoroacetate:

Typical heptane sulfonates include from one to forty molecules of heptane sulfonate. Preferably, each branch is bound to a conjugate of twelve molecules of heptane sulfonate, respectively. The preferred conjugate is forty-eight molecules of heptane sulfonate:

Compound b Typical trifluoroacetate salts include from one to twelve molecules of trifluoroacetate. Preferably, each branch is bound to a conjugate of three molecules of trifluoroacetate, respectively. The preferred conjugate is twelve molecules of trifluoroacetate:

Typical heptane sulfonates include from one to twelve molecules of heptane sulfonate. Preferably, each branch is bound to a conjugate of three molecules of heptane sulfonate, respectively. The preferred conjugate is twelve molecules of heptane sulfonate:

The typical trifluoroacetate salt of compound c comprises from one to twelve molecules of trifluoroacetate, preferably a conjugate of three molecules of trifluoroacetate per molecule, respectively. The preferred conjugate is twelve molecules. Trifluoroacetate:

Solid tumor types suitable for compound a include lymphoid, breast, pancreas, ovary, colon, kidney, bile duct, lung, stomach, brain malignant sarcoma, cancer, and are particularly suitable for treating glioma and breast cancer brain metastasis.

Solid tumor types for compounds b and c include ovarian cancer, breast cancer, lung cancer, endometrial cancer, brain tumor, mesothelioma, kidney cancer, stomach cancer, head and neck cancer, lung cancer, colorectal cancer, testicular cancer, Especially suitable for ovarian cancer, breast cancer and lung cancer.

Specific embodiment

The invention will be described in detail below. However, the invention may be embodied in many different forms and it should not be limited to the embodiments described herein. The purpose of providing these embodiments is to make the disclosure more complete and comprehensive. The reagents and starting materials used are all commercially available except for the preparation methods. 4armPEG20K-SCM was purchased from Beijing Keykai Technology Co., Ltd. and its molecular weight is about 20kDa. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

The structure of 4arm-PEG20K-SCM is as follows:

Glossary

Example 1

Preparation of BP103a01

Under a nitrogen atmosphere, add 200 mL of pyridine, 120 g of BP103a00 (1.0 eq) to a 1000 ml three-necked flask, stir to cool to 0 ° C, add 151.8 g of TsCl (1.0 eq) in portions, stir for 1 h, then slowly warm to room temperature, continue stirring 3- 4h. After completion of the reaction, the reaction solution was poured into ice-dilute hydrochloric acid solution, extracted with EA was added, EA layer was washed once with dilute hydrochloric acid, saturated sodium bicarbonate, washed with brine, dried over anhydrous Na ₂ SO _4, the solvent was distilled off under reduced pressure The silica gel column chromatography obtained 55 g of pure BP103a01.

Preparation of BP103a02

Add 55 g of BP103a01 (1.0 eq) and 160 mL of DMSO to a 1000 mL three-necked flask, stir well, then add 23.52 g (2.0 eq) of NaN ₃ , heat to 50 ° C for 3 hours, cool to room temperature, and pour the reaction solution into water. The organic phase was combined, dried over anhydrous sodium sulfate and concentrated to give a crystals.

Preparation of BP103a03

To a 1 L hydrogenation reactor, 29 g of BP103a02, methanol (360 mL), palladium carbon (5.0 g) were added, stirred, replaced with nitrogen, and subjected to hydrogen reaction for 3-4 hours. After the reaction was monitored by TLC, the reaction mixture was filtered, and the filtrate was concentrated to give an oil of BP103a03. g.

Preparation of BP103a04

23.5 g of compound BP103a03 (1.0 eq), 68.6 g (Boc) ₂ O (2.0 eq), and 500 ml of a mixed solution of methanol:triethylamine (9:1) were added to a 1 L three-necked flask, and the mixture was stirred and heated to reflux for 1 h. After the reaction was completed by TLC, methanol (methanol) was evaporated, dissolved in water, and extracted three times with dichloromethane. The organic layer was washed with water, dried over anhydrous sodium sulfate, and evaporated to dryness to afford 34.8 g of solid BP103a04.

Preparation of BP103a05

34.8 g of compound BP103a04 (1.0 eq), 150 ml of toluene and THF, 58.2 g (3 eq) of bromoacetic acid were added to a 1000 mL three-necked flask, stirred, heated to 45-50 ° C, and then added with 33.5 g of sodium hydroxide (6 eq). After overnight, the reaction was completed by TLC. The reaction mixture was evaporated, water and EA was evaporated, and the aqueous phase was adjusted to pH 3. The aqueous phase was extracted with methylene chloride. The methylene chloride layer was combined, dried over anhydrous sodium sulfate and concentrated to yield Compound 18g.

Preparation of BP103a

18 g of compound BP103a05, 100 ml of EA was added to a 250 mL three-necked flask, stirred and dissolved, and then cooled to 0 ° C. 150 ml of EA/HCl (3.5 M) was added, and the temperature was maintained at 0 ° C. The reaction was monitored by TLC, filtered, and the filter cake was washed with TBME to give a white solid. BP103a 10.4g.

Preparation of Compound 2

To a 100 mL flask, 3.0 g of BP103a (1.0 eq), Compound 1 4.0 g (1.0 eq), 40 ml of DCM, 4.0 ml of DIEA (2.0 eq) was stirred at room temperature, and the reaction was completed by TLC. Oil-like compound 2 5.2 g.

Preparation of Compound 3

9.00 g of compound 2 (1.0 eq), 3.96 g of HOSU (1.53 eq), 90 ml of DCM, 6.60 g of EDC·HCl (1.53 eq) were added to a 200 mL three-necked flask, and reacted at room temperature for 2 h. After completion of TLC monitoring, the pH was diluted with DCM. The aqueous solution of 50 mmol/L of potassium dihydrogen phosphate was washed twice with saturated aqueous sodium chloride solution, washed with saturated brine, dried over anhydrous sodium sulfate and evaporated.

Preparation of Compound 4

To a 200 mL flask was added 2.93 g of compound NH ₂ -Lys(Boc)-OH (1.0 eq), 60 ml of water, 2.00 g of NaHCO ₃ (2.0 eq), stirred, and 5.9 g of compound 3 (1.0 eq) was added dropwise in 60 ml of DME. To the solution, 60 ml of THF was added, and the mixture was stirred overnight. The mixture was evaporated to dryness.

Preparation of compound 6

To a 250 mL round bottom flask, 3.50 g of compound 5 (1.0 eq), 52.5 ml of DMF was added, and the mixture was heated to 60 ° C to dissolve. After 5-10 min, DMF was distilled off under reduced pressure, and 300 ml of n-heptane was added for distillation under reduced pressure, three times, and dried. Then, 105 ml of DCM, 1.08 g of Boc-Gly-OH (1.2 eq), 63 mg of DMAP (0.1 eq), and a solution of 1.59 g of DCC (1.5 eq) dissolved in 10 ml of DCM were added dropwise, and reacted at 20 ° C for 4 hours, and the reaction was monitored by TLC. After filtration, the mixture was concentrated to a remaining 25% by volume, 120 ml of IPA was added, 75% of the solvent was distilled off, 150 ml of n-heptane was added, and the mixture was stirred at room temperature for 1 hour, filtered, washed twice with n-heptane, and dried to give a pale yellow solid. 6.

Preparation of Compound 7

To a 100 mL three-necked flask, 4.02 g of compound 6, 50 ml of DCM was added, stirred and dissolved, and 11.6 ml of TFA was added dropwise, and reacted at room temperature for 2 h. After completion of TLC monitoring, 150 ml of acetonitrile was added, 120 ml of solvent was distilled under reduced pressure, and poured into 320 ml of TBME solution, and stirred. After 30 min, filtered, the filter cake was washed with EtOAc EtOAc.

Preparation of Compound 8

To a 200 mL three-necked flask was added 3.69 g of Compound 7, 100 ml of DCM, 3.21 g (1.05 eq) of Compound 4, 2.7 ml of DIEA (3.0 eq), 1.2 ml of DEPC (1.5 eq), and reacted at room temperature for 4 h. After TLC monitoring reaction, DCM After diluting, it was washed twice with water, washed with saturated brine, dried, concentrated, and purified by HPLC and then lyophilized to give a pale yellow solid compound 8 1.85 g.

Preparation of compound 9

To a 50 mL round bottom flask, 260 mg of compound 8 and 10 ml of 20% TFA/DCM were added, and the mixture was reacted at room temperature for 4 h. After completion of the reaction by TLC, the mixture was poured into TBME, centrifuged, dried and purified by HPLC to obtain a pale yellow solid compound 9 210 mg.

Preparation of Compound 10

To a 10 mL round bottom flask was added 51 mg of compound 9 (4.0 eq), 2 ml of DCM, 11 ul of TEA (8.0 eq), 201 mg of 4arm PEG 20K-SCM (1.0 eq), allowed to react overnight at room temperature, concentrated, added to TBME, centrifuged, and prepared by HPLC. Purified and lyophilized to give a yellow-green solid compound 10 85 mg.

Preparation of PP04a

The synthesis of PP04a is carried out by solid phase synthesis using Fmoc method well known to the skilled person. 2-Cl-Trt Resin is used, Fmoc is removed by using 20% piperidine/DMF, and the coupling reagent is HOBT/DIC, DMF is used as the reaction solvent, and the reaction monitoring is carried out. The following protected amino acids were attached to the resin by ninhydrin assay: Fmoc-Cys(Trt)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Lys (Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH , Fmoc-Leu-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc -Arg(Pbf)-OH, Fmoc-Leu-OH, DMF washing, methanol washing, washing with DCM, drying, adding a cleavage reagent (TFA: thioanisole: phenol: TIS = 85:5:5:5), reaction After 2 hours, it was precipitated with ice TBME and centrifuged to obtain crude PP04a, which was purified by HPLC and then freeze-dried to obtain pure PP04a.

To a 10 mL round bottom flask was added 500 mg of compound 10 (1.0 eq), 10 ml of pH=7, 0.01 M PBS, and after dissolution, 197 mg (4.0 eq) of PP04a was dissolved in 5 ml of a solution of pH=7, 0.01 M in PBS, room temperature. After reacting for 4 hours, dialysis and concentration to obtain compound 11 (ie, compound a) as described above, dissolving the crude product in methanol, adding TFA to adjust pH=5-6, concentrating, adding to TBME, solid precipitation, centrifugation, and drying to obtain yellow solid compound 12 462 mg.

Compound 11 was purified by reverse-phase HPLC (silica gel: C18, 300A; mobile phase: sodium heptanesulfonate/water, acetonitrile). After the pure product was collected, pH was adjusted to 4 to 5, and then desalted by reverse HPLC (silica gel: C18) , 300A; mobile phase: acetic acid / water, acetonitrile), the pure product was collected, concentrated to remove the organic solvent, and lyophilized to obtain a white powder-like compound 13.

Compound 11 MALDI-TOF detected a molecular weight of 34,500.78.

The molecular weight of Compound 12 MALDI-TOF was 34,451.86.

Compound 13 MALDI-TOF detected a molecular weight of 34,552.21.

Example 2

Synthesis of CDDRD-folic acid

The synthesis of the polypeptide CDDRD-folic acid is carried out by solid phase synthesis using Fmoc method well known to the skilled person. 2-Cl-Trt Resin is used, Fmoc is removed by using 20% piperidine/DMF, and the coupling reagent is HOBT/DIC, DMF is used for the reaction. Solvent, reaction monitoring using ninhydrin detection method, the following protective amino acids were sequentially attached to the resin: Fmoc-Cys(Trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc- Arg(Pbf)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Glu-OtBu, trifluoroacetyl pteroic acid, DMF washed with hydrazine hydrate to remove trifluoroacetyl group, DMF washing, methanol washing, DCM After washing, it was dried, and a cleavage reagent (TFA: thioanisole: phenol: TIS=85:5:5:5) was added. After reacting for 2 hours, it was precipitated with ice TBME and centrifuged to obtain a crude product which was purified by HPLC and then freeze-dried. Pure.

To a 10 mL round bottom flask was added 500 mg of compound 10 (1.0 eq), 10 ml of pH=7, 0.01 M PBS, and dissolved for use; another 83 mg (4.0 eq) of CDDRD-folic acid was added, 1 ml of purified water was added, and 10 mmol was used. /L aqueous sodium hydrogencarbonate solution adjusted to pH = 7, this solution was added to the solution of compound 10 for 4 hours at room temperature, dialysis, concentration, to obtain compound 14 (the compound b described above), dissolved in methanol, added to TFA to adjust The mixture was concentrated, added to TBME, and solid was precipitated, centrifuged, and dried to give a yellow solid compound 15 367 mg.

After purification by reverse-phase HPLC (silica gel: C18, 300A; mobile phase: sodium heptanesulfonate/water, acetonitrile), the pure product was collected, pH was adjusted to 4 to 5, and then desalted by reverse-phase HPLC (silica gel: C18) , 300A; mobile phase: acetic acid / water, acetonitrile), the pure product was collected, concentrated to remove the organic solvent, and lyophilized to obtain a white powder-like compound 16.

The molecular weight of the compound 14 MALDI-TOF was 28,887.03.

Compound 15 MALDI-TOF detected a molecular weight of 28920.25.

The molecular weight of Compound 16 MALDI-TOF was 28,951.38.

Example 3

Preparation of compound 17

Solid phase synthesis by Fmoc method well known to the skilled person, 2-Cl-Trt Resin, removal of Fmoc by 20% piperidine/DMF, coupling reagent using HOBT/DIC, DMF as reaction solvent, reaction monitoring using 茚三A ketone assay in which the following protected amino acids are attached to the resin: Fmoc-Lys(Boc)-OH, Fmoc-PGE3-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Glu-OtBu, trifluoroacetyl pteroic acid, DMF washed with hydrazine hydrate to remove trifluoroacetyl group, DMF washing, methanol washing, DCM washing After drying, a cleavage reagent (TFA: thioanisole: phenol: TIS=85:5:5:5) was added, and after reacting for 2 hours, it was precipitated with ice TBME and centrifuged to obtain a crude product which was purified by HPLC and then freeze-dried to obtain a compound. 17 pure products.

Preparation of Compound 19

4 g of compound 17 (1.0 eq), 2.32 g of compound 18 (1.0 eq), 56 ml of DMF were added to a 100 mL three-necked flask, stirred and cooled to 0 ° C, then 1.19 g DIEA (3.0 eq) was added, 1.47 g TBTU (1.5 eq. After the reaction was carried out for 2 hours at room temperature, the HPLC reaction was completed, and the reaction mixture was poured into TBME to precipitate a solid, which was filtered and dried to give 7.67 g of a yellow solid.

Preparation of Compound 20

To a 100 mL three-necked flask, 7.05 g of Compound 19 was added, and a cleavage reagent (TFA: thioanisole: phenol: TIS = 85:5:5:5) was added, and after reacting for 2 hours, it was precipitated with ice TBME and centrifuged to obtain a crude product. The HPLC preparation was purified and lyophilized to give 1.36 g of pure product 20.

Preparation of Compound 21 (ie, Compound c as described above)

0.58 g of compound 20 (4.0 eq), 1.31 g of 4arm-PEG20K-SCM (1.0 eq), 13 ml of DMF, 84 mg of DIEA were added to a 10 mL round bottom flask, and after HPLC reaction, the mixture was poured into TBME, and the solid was precipitated to obtain a compound. 21 crude product 1.82g.

Preparation of Compound 22

Compound 21, methanol was dissolved in crude product, added to TFA to adjust pH = 5-6, concentrated, added to TBME, solid precipitated, centrifuged, and dried to give compound 22 as a yellow solid.

Preparation of Compound 23

Compound 21 was purified by reverse-phase HPLC (silica gel: C18, 300A; mobile phase: sodium heptanesulfonate/water, acetonitrile). After the pure product was collected, pH was adjusted to 4 to 5, and then desalted by reverse HPLC (silica gel: C18) , 300A; mobile phase: acetic acid / water, acetonitrile), the pure product was collected, concentrated to remove the organic solvent, and lyophilized to obtain a white powder-like compound 23.

The molecular weight of the compound 21 MALDI-TOF was 27,328.05.

Compound 22 MALDI-TOF detected a molecular weight of 27392.54.

Compound 23 MALDI-TOF detected a molecular weight of 27419.56.

Example 4 Effect of Compound a on Survival Rate of Brain In situ Model of U87MG Nude Mice

Test samples: irinotecan, nktr-102, compound a.

Among them, the preparation method of nktr-102 is as follows:

Compound 7 (829 mg, 4.5 eq) in the Example was added to a 250 mL reaction flask, DCM (50 mL), triethylamine (221 mg, 9.0 eq) was added, dissolved and then added 4arm-PEG20K-SCM (5.00 g, 1.0) Eq) was added to the reaction flask. After the HPLC monitoring reaction did not progress significantly, about 20 mL of DCM was distilled off under reduced pressure. The solution was poured into 300 mL of TBME and stirred to precipitate. After filtration, 5.4 g of crude product was obtained. The crude product was purified by HPLC, desalted, and adjusted to pH 5-6 with dilute hydrochloric acid. Lyophilized to give 2.71 g of pale green powder nktr-102.

Reagents: RPMI-1640 medium, trypsin, cyan-chain double antibody, saline.

Experimental animals: Female BALB/c nude mice (only: 60; weeks of age: 6-8 weeks) purchased from Beijing Vital Lihua Experimental Animal Technology Co., Ltd., raised in SPF animal room, temperature 20 ~ 25 ° C, Relative humidity 40% ~ 70%, light and dark lighting for 12 hours; animals free access to water and feeding. After about 1 week of normal feeding, veterinary examination, mice with good signs of good condition can be included in this experiment. Before the grouping, use the marker to mark the root of the animal. After grouping, each animal is identified by ear clipping.

Transplanted tumor tumor strain: glioma cell line U87MG, derived from the Cell Bank of the Typical Culture Collection Committee of the Chinese Academy of Sciences (CAS, laboratory laboratory liquid nitrogen cryopreservation).

experimental method:

NCI-N87 cell culture: NCI-N87 cells were cultured in RPMI-1640 medium in 5% CO ₂ at 37 ° C; digested with 0.25% trypsin; passaged weekly according to cell growth 1 to 2 times, the passage ratio is 1:2 to 1:6.

Animal model preparation: NCI-N87 cells were collected in logarithmic growth phase. After cell counting, resuspend in serum-free RPMI-1640 medium, adjust the cell concentration to 1×10 ⁸ cells/mL; pipette the cells with a pipette. After dispersing evenly, it was placed in a 50 mL centrifuge tube, and the centrifuge tube was placed in an ice box; the cell suspension was aspirated with a 1 mL syringe, and the human glioma cell line U87MG was cultured in vitro by microinjection using the guidance of an animal stereotactic instrument. 1 μL (1×10 ⁵ cells/cell) was used to establish an in situ model of U87MG glioma, and the state of the animals was observed periodically after inoculation. On the 12th day after inoculation, 66 animals were selected and divided into 4 groups (n=6) by randomized block method.

Formulation of irinotecan preparation: Weigh 12.0 mg of irinotecan, add 0.15 mL of 1% lactic acid, vortex to completely dissolve the drug, and then add 2.85 mL of 1% sorbitol aqueous solution separately, vortex and mix. Evenly, the ratio of 1% lactic acid to 1% sorbitol aqueous solution in the solution was about 5:95 (v/v). The free form concentration of irinotecan in the solution was 4.0 mg·mL ^-1 .

Preparation of nktr-102 preparation: Before each administration, accurately weigh 101.5mg of nktr-102, add 2.5mL of normal saline, vortex to completely dissolve the drug, and the concentration of irinotecan in the solution is 4.0mg. ·mL ^-1 .

Compound a administration preparation preparation: accurately weighed, added 2.5 mL of physiological saline, vortexed to completely dissolve the drug, and the concentration of irinotecan was 4.0 mg·mL ^-1 .

Animal grouping and administration: The first administration was started on the day of grouping, and the administration volume was 10 mL·kg ^-1 . The first group was the solvent control group, and the blank solvent was administered by tail vein injection once every 4 days for a total of 3 times (Q4D×3). Groups 2-4 were administered with irinotecan, nktr-102, and compound a, respectively, at a dose of 40 mg·kg ^-1 (calculated as irinotecan), Q4D × 3, respectively.

Animal survival was recorded and the median survival time was calculated. The survival rate difference was compared using the Log Rank time series test. The statistical process was performed using SPSS 16.0 statistical software, and the median survival time was calculated using graphpad 5.0 software. The results are shown in Table 1.

Table 1 Animal survival time (days)

*Compared with the median survival time of the blank solvent, irinotecan and nktr-102 groups, P<0.05

The experimental results show that compound a has a good inhibitory effect on glioma, which can significantly prolong the median survival time of animals, and is superior to irinotecan and nktr-102.

Example 5 Inhibition of Compound A on Human Breast Cancer MDA-MB-231 Brain Metastasis Model

Test samples: irinotecan, nktr-102, compound a.

Reagents: RPMI-1640 medium, trypsin, cyan-chain double antibody, saline.

Transplanted tumor tumor strain: Human breast cancer MDA-MB-231-Luc was purchased from the Institute of Cell Biology, Shanghai Institute of Chinese Academy of Sciences.

experimental method:

MDA-MB-231-Luc was cultured in DMEM medium (GIBCO, USA) containing 10% fetal bovine serum FBS (GIBCO, USA) and cultured in a 37 ° C incubator containing 5% CO ₂ .

Brain metastasis MDA-MB-231-Luc cell preparation: MDA-MB-231-Luc cells in logarithmic growth phase were collected, cell counted and resuspended in serum-free RPMI-1640 medium, and heart injected with 5×10 ⁵ cells . Three to four weeks after cardiac injection, brain metastatic nude mice were selected by bioimaging and brain metastatic MDA-MB-231-Luc cells were isolated. The isolated MDA-MB-231-Luc cells were inoculated into the nude mouse heart, brain-transferred nude mice were selected by bioimaging, and brain metastatic MDA-MB-231-Luc cells were isolated. This was repeated 8 times until no MDA-MB-231-Luc cells were transferred in other tissues.

For the preparation of the test article, see Example 4.

Animal grouping and administration: Animal hearts were inoculated with previously prepared brain metastasis MDA-MB-231-Luc cells 5 x 10 ⁵ /only. The brain metastasis was confirmed by bioimaging 21 days after the inoculation, and the nude mice which confirmed the brain metastasis were selected and the first administration was started on the day of grouping, and the administration volume was 10 mL·kg ^-1 . The first group was the solvent control group, and the blank solvent was administered by tail vein injection once every 4 days for a total of 3 times (Q4D×3). Groups 2-4 were administered with irinotecan, nktr-102, and compound a, respectively, at a dose of 40 mg·kg ^-1 (calculated as irinotecan), Q4D × 3, respectively.

Animal survival was recorded and the median survival time was calculated. The survival rate difference was compared using the Log Rank time series test. The statistical process was performed using SPSS 16.0 statistical software, and the median survival time was calculated using graphpad 5.0 software. The results are shown in Table 2.

Table 2 Animal survival time (days)

The results showed that compound a had a good inhibitory effect on MDA-MB-231-Luc brain metastasis, which significantly prolonged the median survival time of animals and was superior to irinotecan and nktr-102.

Example 6 Antitumor efficacy test of compound b against human ovarian cancer SK-OV-3 xenograft tumor in nude mice

Test samples: irinotecan, nktr-102, compound b.

Reagents: McCoy's 5A medium, fetal bovine serum (FBS), trypsin, cyan-chain double antibody, physiological saline.

Experimental animals: Female BALB/c nude mice (only: 150; weekly age: 6-8 weeks) purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., and raised in SPF animal room of Suzhou Shengsu New Drug Development Co., Ltd. , temperature 20 ~ 25 ° C, relative humidity 40% ~ 70%, light and dark lighting for 12 hours; animals free access to water and feeding. After about 1 week of normal feeding, veterinary examination, mice with good signs of good condition can be included in this experiment. Before the grouping, use the marker to mark the root of the animal. After grouping, each animal is identified by ear clipping.

Transplanted tumor tumor strain: human ovarian cancer SK-OV-3, derived from the Cell Bank of the Typical Culture Collection Committee of the Chinese Academy of Sciences (CAS, laboratory laboratory liquid nitrogen cryopreservation).

experimental method

Cell culture: SK-OV-3 was cultured in McCoy's 5A medium (GIBCO, USA) containing 10% fetal bovine serum FBS (GIBCO, USA) and cultured in a 37 ° C incubator containing 5% CO ₂ .

Animal model preparation: cell inoculation method to establish a subcutaneous transplantation model of tumor nude mice: collect tumor cells in logarithmic growth phase, count and resuspend in 1×PBS, adjust the cell suspension concentration to 5×10 ⁷ /ml and compare with Matrigel 1:1 Mix well. Tumor cells were inoculated subcutaneously in the right side of nude mice with a 1 ml syringe (4 gauge needle), 5 x 10 ⁶ /mouse, and 50 animals were inoculated. When the tumor volume reached 100-300 mm ³ , the animals were randomly grouped by random block method. After the start of the experiment, the tumor diameter was measured twice a week, the tumor volume was calculated, and the animal weight was weighed and recorded.

Solvent preparation: Weigh 0.5g of sorbitol into a 50mL centrifuge tube, add 50mL of water for injection to the centrifuge tube, vortex to completely dissolve the solid substance, and prepare a 1% concentration of sorbitol aqueous solution (w/v) Store in a refrigerator at 4 °C.

For the preparation of the test article for administration, referring to Example 4, the concentration of irinotecan in the compound b solution was 4.0 mg·mL ^-1 .

Animal grouping and administration: The first administration was started on the day of grouping, and the experiment was terminated after 21 days, and the administration volume was 10 mL·kg ^-1 . The first group was the solvent control group, and the blank solvent was administered by tail vein injection once every 4 days for a total of 3 times (Q4D×3). Groups 2-4 were administered with irinotecan, nktr-102, and compound b in the tail vein, respectively, at a dose of 40 mg·kg ^-1 (calculated as irinotecan).

After the end of the experiment, the animals were weighed and the tumor diameter was measured and the animals were euthanized (CO ₂ ). Tumor tissue was stripped and weighed to calculate the tumor inhibition rate.

The tumor volume (TV) is calculated as follows:

TV(mm ³ )=l×w ² /2

Wherein, l represents the tumor long diameter (mm); w represents the tumor short diameter (mm).

The relative tumor volume (RTV) is calculated as:

RTV=TV _t /TV _initial

Wherein TV _initial is the tumor volume measured at the time of group administration; TV _t is the tumor volume at each measurement during administration.

The relative tumor growth rate (%T/C) is calculated as:

%T/C=100%×(RTV _T /RTV _C )

Among them, RTV _T represents the treatment group RTV; RTV _C represents the solvent control group RTV.

Statistical analysis method: The test data was calculated by Microsoft Office Excel 2007 software and related statistical processing. Data were expressed as mean ± standard error (Mean ± SE) unless otherwise specified, and t-test was used for comparison between the two groups.

For the human cancer xenograft model, the relative tumor proliferation rate T/C (%) is recommended as the experimental evaluation index. The lower the proliferation rate, the better the tumor suppression effect. The results are shown in Table 3.

Table 3 Compound b on tumor proliferation rate T / C (%)

*P<0.05 compared with RTV in blank solvent, irinotecan and nktr-102 groups

#Compared with %T/C of blank solvent, irinotecan and nktr-102 group, P<0.05

The results showed that compound b had a good inhibitory effect on tumor growth of human ovarian cancer SK-OV-3 cell line xenograft model and was superior to irinotecan and nktr-102.

Example 7 Antitumor efficacy test of compound c against human ovarian cancer SK-OV-3 xenograft tumor in nude mice

The test method is the same as that in Example 6. The results are shown in Table 4:

Table 4 Compound c on tumor proliferation rate T/C (%)

*P<0.05 compared with RTV in blank solvent, irinotecan and nktr-102 groups

#Compared with %T/C of blank solvent, irinotecan and nktr-102 group, P<0.05

The results showed that compound c had a good inhibitory effect on tumor growth of human ovarian cancer SK-OV-3 cell line xenograft model and was superior to irinotecan and nktr-102.

Example 8 Antitumor efficacy test of compound b on human breast cancer MDA-MB-231 xenograft tumor in nude mice

Test samples: irinotecan, nktr-102, compound b.

Reagents: DMEM medium, fetal bovine serum (FBS), trypsin, cyan-chain double antibody, saline.

Transplanted tumor tumor strain: human breast cancer MDA-MB-231, derived from the Cell Bank of the Typical Culture Collection Committee of the Chinese Academy of Sciences (CAS, laboratory laboratory liquid nitrogen cryopreservation).

experimental method

Cell culture: MDA-MB-231 was cultured in DMEM medium (DMEM, USA) containing 10% fetal calf serum FBS (GIBCO, USA) and cultured in a 37 ° C incubator containing 5% CO 2 .

Animal model preparation Cell inoculation method To establish a subcutaneous transplantation model of tumor nude mice: tumor cells in logarithmic growth phase were collected, counted, resuspended in 1×PBS, and the cell suspension concentration was adjusted to 1×10 ⁷ /ml. Tumor cells were inoculated subcutaneously in the right side of nude mice with a 1 ml syringe (4 gauge needle), 1 x 10 ⁶ /mouse, and 50 animals were inoculated. When the tumor volume reached 100-300 mm ³ , the animals were randomly grouped by random block method. After the start of the experiment, the tumor diameter was measured twice a week, the tumor volume was calculated, and the animal weight was weighed and recorded.

For the preparation method of the test article for administration, see Example 4, and the concentration of irinotecan in the compound b solution was 4.0 mg·mL ^-1 .

After the end of the experiment, the animals were weighed and the tumor diameter was measured and the animals were euthanized (CO ₂ ). The tumor tissue was stripped and weighed, and the tumor weight inhibition rate and the tumor inhibition rate were calculated in the same manner as in Example 6. The results are shown in Table 5.

Table 5 Compound b on tumor proliferation rate T / C (%)

*P<0.05 compared with RTV in blank solvent, irinotecan and nktr-102 groups

#Compared with %T/C of blank solvent, irinotecan and nktr-102 group, P<0.05

The results showed that compound b had a good inhibitory effect on tumor growth of human breast cancer MDA-MB-231 cell line xenograft model and was superior to irinotecan and nktr-102.

Example 9 Antitumor efficacy test of compound c on human breast cancer MDA-MB-231 xenograft tumor in nude mice

The test method is the same as that in Example 8. The results are shown in Table 6:

Table 6 Compound c on tumor proliferation rate T/C (%)

*P<0.05 compared with RTV in blank solvent, irinotecan and nktr-102 groups

#Compared with %T/C of blank solvent, irinotecan and nktr-102 group, P<0.05

The results showed that compound c had a good inhibitory effect on tumor growth of human breast cancer MDA-MB-231 cell line xenograft model and was superior to irinotecan and nktr-102.

Example 10 Antitumor efficacy test of compound b on human lung cancer SPC-A-1 xenograft tumor in nude mice

Test samples: irinotecan, nktr-102, compound b.

Reagents: RPMI-1640 medium, fetal bovine serum (FBS), trypsin, cyan-chain double antibody, saline.

Transplanted tumor tumor strain: human lung cancer SPC-A-1, derived from the Cell Bank of the Typical Culture Collection Committee of the Chinese Academy of Sciences (CAS, laboratory laboratory liquid nitrogen cryopreservation).

experimental method

Cell culture: SPC-A-1 was cultured in RPMI-1640 medium containing 10% fetal bovine serum FBS (GIBCO, USA) and cultured in a 37 ° C incubator containing 5% CO 2 .

Animal model preparation: Cell inoculation method was established to establish a subcutaneous transplantation model of tumor nude mice: tumor cells in logarithmic growth phase were collected, counted, resuspended in 1×PBS, and the cell suspension concentration was adjusted to 2×10 ⁷ /ml. Tumor cells were inoculated subcutaneously in the right side of nude mice with a 1 ml syringe (4 gauge needle), 2 x 10 ⁶ /mouse, and 50 animals were inoculated. When the tumor volume reached 100-300 mm ³ , the animals were randomly grouped by random block method. After the start of the experiment, the tumor diameter was measured twice a week, the tumor volume was calculated, and the animal weight was weighed and recorded.

After the end of the experiment, the animals were weighed and the tumor diameter was measured and the animals were euthanized (CO ₂ ). The tumor tissue was stripped and weighed, and the tumor weight inhibition rate and the tumor inhibition rate were calculated in the same manner as in Example 7. The results are shown in Table 7.

Table 7 Compound b on tumor proliferation rate T/C (%)

*P<0.05 compared with RTV in blank solvent, irinotecan and nktr-102 groups

#Compared with %T/C of blank solvent, irinotecan and nktr-102 group, P<0.05

The results showed that compound b had a good inhibitory effect on tumor growth of human lung cancer SPC-A-1 cell line xenograft model and was superior to irinotecan and nktr-102.

Example 11 Antitumor efficacy test of compound c on human lung cancer SPC-A-1 xenograft tumor in nude mice

The test method is the same as that in Example 10. The results are shown in Table 8:

Table 8 Compound c on tumor proliferation rate T/C (%)

*P<0.05 compared with RTV in blank solvent, irinotecan and nktr-102 groups

#Compared with %T/C of blank solvent, irinotecan and nktr-102 group, P<0.05

The results showed that compound c had a good inhibitory effect on tumor growth of human lung cancer SPC-A-1 cell line xenograft model and was superior to irinotecan and nktr-102.

Claims

a multi-branched drug conjugate having the following structural formula (IV) or a pharmaceutically acceptable salt thereof,

Where R is the organic center
q is 4;

POLY is polyethylene glycol
n is 113, and the oxygen atom labeled "&" is an atom connected to the organic center "R";

Representing the junction of atoms;

L is a multivalent linker; T is a targeting molecule selected from the group consisting of LRKLRKRLLLRKLRKRLL and folic acid; and D is irinotecan.
The multi-branched drug conjugate of claim 1 or a pharmaceutically acceptable salt thereof, wherein

L is

The symbol "*" represents the point of attachment of the multivalent linker L to the target molecule T by "cysteine", "#" represents the point of attachment of the multivalent linker L to the active agent D, and "%" represents the link of the multivalent linker L and POLY. Junction,

T is LRKLRKRLLLRKLRKRLL,

That is, the compound represented by formula (I)
The multi-branched drug conjugate of claim 1 or a pharmaceutically acceptable salt thereof, wherein

L is

The symbol "*" represents the point of attachment of the multivalent linker L to the target molecule T by "cysteine", "#" represents the point of attachment of the multivalent linker L to the active agent D, and "%" represents the link of the multivalent linker L and POLY. Junction,

T is folic acid,

That is, the compound represented by formula (II)
The multi-branched drug conjugate of claim 1 or a pharmaceutically acceptable salt thereof, wherein

L is

The symbol "*" represents the point of attachment of the multivalent linker L to the target molecule T by "cysteine", "#" represents the point of attachment of the multivalent linker L to the active agent D, and "%" represents the link of the multivalent linker L and POLY. Junction,

T is folic acid,

That is, the compound represented by formula (III)
The multi-branched drug conjugate according to claim 1 or a pharmaceutically acceptable salt thereof, wherein the pharmaceutically acceptable salt comprises nitrate, sulfate, phosphate, hydrofluoric acid, hydrochloric acid Salt, hydrobromide, hydroiodide, formate, lactate, benzoate, acetate, trifluoroacetate, dichloroacetate, trichloroacetate, mixed chlorine Fluoroacetate, citrate, oxalate, sulfonate, methanesulfonate, triflate, heptane sulfonate.
The multi-branched drug conjugate according to claim 2 or a pharmaceutically acceptable salt thereof, wherein the salt of the conjugate is forty-eight molecules of trifluoroacetic acid each of which is bound to a twelve-molecular salt per branch salt:

Forty-eighth molecules of heptane sulfonate combined with twelve molecular salts per branch:
The multi-branched drug conjugate according to claim 3 or a pharmaceutically acceptable salt thereof, wherein the salt of the conjugate is twelve molecules of trifluoroacetic acid each of which binds to a three-molecular salt per branch salt:

Twelve molecules of heptane sulfonate combined with three molecules of salt per branch:
The multi-branched drug conjugate according to claim 4 or a pharmaceutically acceptable salt thereof, wherein the salt of the conjugate is twelve molecules of trifluoroacetic acid each of which binds to a three-molecular salt per branch salt:

Twelve molecules of heptane sulfonate combined with three molecules of salt per branch:
A method for preparing a multi-branched drug conjugate according to claim 2 or a pharmaceutically acceptable salt thereof, comprising:

(1) after reacting irinotecan hydrochloride with Boc-Gly-OH, forming a salt with TFA to obtain compound 7;

(2) Compound 7 is reacted with Compound 4 to give Compound 8;

(3) Compound 8 is reacted with TFA, and reacted with 4arm-PEG20K-SCM to obtain compound 10;

(4) Compound 10 is reacted with PP04a to give the conjugate of formula (I);

The structure of PP04a is as follows:
A method for preparing a multi-branched drug conjugate according to claim 3 or a pharmaceutically acceptable salt thereof, comprising:

Compound 10 is reacted with CDDRD-folic acid to give a conjugate of formula (II).
A method for preparing a multi-branched drug conjugate according to claim 4 or a pharmaceutically acceptable salt thereof, comprising:

(1) compound 17 is reacted with compound 18 to give compound 19;

(2) Compound 19 is salted with TFA and then reacted with 4arm-PEG20K-SCM to obtain a coupling of formula (III).
Use of a multi-branched drug conjugate according to any of claims 1-8, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of cancer.
The multi-branched drug conjugate according to claim 2 or a pharmaceutically acceptable salt thereof for the preparation of a malignant sarcoma for treating lymph, breast, pancreas, ovary, colon, kidney, bile duct, lung, stomach, brain, The application of cancer drugs.
The use according to claim 13, wherein said cancer is glioma and breast cancer brain metastasis.
The multi-branched drug conjugate according to claim 3 or 4, or a pharmaceutically acceptable salt thereof, for use in the treatment of ovarian cancer, breast cancer, lung cancer, endometrial cancer, brain tumor, mesothelioma, kidney Application in drugs for cancer, stomach cancer, head and neck cancer, lung cancer, colorectal cancer, testicular cancer.
A pharmaceutical composition comprising the multi-branched drug conjugate of any of claims 1-8, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.